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The recovered vesicle fractions contain bands at the expected positions for the supplied SNARE proteins. The v-SNARE preparation contains its principal band, and the t-SNARE preparation contains both t-SNARE and the SNAP25 doublet. Thus protein-containing vesicle fractions have been recovered, consistent with successful reconstitution.

The SDS-PAGE experiment does not establish the orientation of the proteins, their proportion facing outward, the stoichiometry in an individual vesicle, or membrane fusion. Nor does a stained band prove that all of its protein is correctly folded and active. Those are separate functional and membrane topology questions.

Lane 4 combines detergent with the absence of the t-SNARE preparation. The lipid bilayer no longer protects inward-facing v-SNARE, and there is no t-SNARE partner to provide complex-dependent protection. Consequently little full-length labelled protein remains. This distinguishes membrane-dependent protection from partner-dependent protection in a protease protection assay. It also helps interpret lane 3: its strong signal requires the partner rather than lipid bilayer enclosure alone.

The N-terminal 1–29 fragment and the 1–15 fragment inhibit invadopodium formation. Their values are around one fifth of the control, and gelatin degradation is strongly reduced too. Full-length t-SNARE, empty vector and the 15–29 fragment remain near control values. The images agree: the inhibitory fragments leave much more intact gelatin beneath the cells. The corresponding published syntaxin-4 study links this phenotype to impaired cargo trafficking.^[ref]